Transmission of many infectious agents by transfusion has dramatically declined due to improved testing methods and careful donor selection (Dodd R Y et al. Transfusion 2002; 42:975-9; Zou S et al. Transfusion 2004; 44:1640-9; Fiebig E W et al. Clin Lab Med 2004; 24:797-823). However, other pathogens for which there are no licensed tests or those emerging pathogens for which we have limited knowledge may pose blood borne infectious risks. The recent outbreak of West Nile virus infection in blood and organ recipients demonstrated the vulnerability of the blood supply to emerging and re-emerging pathogens (Macedo de Oliveira A et al. Transfusion 2004; 44:1695-9; Shepherd J C et al. Am J Transplant 2004; 4:830-3; Pealer L N et al. N Engl J Med 2003; 349: 1236-45). Although the period of high risk to recipients was brief due to the rapid implementation of NAT testing, other agents may not be as amenable to successful test development. Therefore, there is a continued need to investigate broad spectrum inactivation methods for blood components.
One approach to pathogen reduction is treatment using photosensitizing dyes (Horowitz B et al. Blood Cells 1992; 18:141-50′ Lavie G et al. Transfusion 1995; 35:392-400; North J et al. Blood Cells 1992; 18:129-40; Rywkin S et al. Photochem Photobiol 1992; 56:463-9; Wagner S J et al. Transfusion 1992; 33:30-6; Abe H et al. Photochem Photobiol 1995; 61, 402-9; Skripchenko A et al. Photochem Photobiol 1997; 65:451-455; Abe H et al. Photochem Photobiol 1997; 65:873-876; Wagner S J et al. Photochem Photobiol 67:343-349, 1998; Wagner S J et al. Transfusion 38:729-737, 1998; Besselink G A et al. Transfusion 2002 June; 42:728-33; Wagner S et al. Transfusion 2002; 42:1200-1205; Wagner S J et al. Photochem Photobiol 2002; 76:514-517). Although some investigators have studied photosensitizers which bind to viral membranes or capsids for pathogen reduction in red blood cells (RBCs), we have focused our efforts on utilizing nucleic acid binding dyes because this approach, in theory, specifically targets pathogens while leaving anucleate RBCs intact. Unfortunately, RBCs phototreated with these dyes suffer damage from reactive oxygen species generated by photosensitizer bound to the erythrocyte membrane as well as photosensitizer free in solution (Besselink G A et al. Transfusion 2002 June; 42:728-33; Wagner S et al. Transfusion 2002; 42:1200-1205; Wagner S J et al. Photochem Photobiol 2002; 76:514-517). Therefore, nucleic acid binding does not necessarily imply that a candidate dye has no affinity to RBC proteins, glycoproteins or lipids, or that an inconsequential level of dye remains free in solution.
In a previous study, we found that the nucleic acid intercalating dye, dimethylmethylene blue, also bound to RBC membranes. Levels of RBC-bound dye could be diminished by the addition of a competitive membrane binding inhibitor, such as quinacrine, whose structure is similar to that of the sensitizer. Using quinacrine, photo-induced hemolysis from dimethylmethylene blue could be significantly reduced under conditions that maintained >6 log10 reduction of model viruses (Wagner S J et al. Photochem Photobiol 2002; 76:514-517). However, hemolysis from phototreated cells protected by the competitive inhibitor remained, possibly from photodynamic action of free dye in solution.
In order to minimize red cell damage from reactive oxygen species emanating from free dye, we investigated the use of a novel nucleic acid intercalating photosensitizer whose flexible structure is only active when dye is rigidly bound to substrate. One such flexible dye is thiopyrylium (TP), which cannot generate reactive oxygen species when free in solution because the energy from absorbed light can be dissipated through bond rotation (Wagner S J et al. Transfusion 2005; 45:752-60). When flexible dyes like TP are rigidly bound to substrate such as nucleic acid, the lifetime of their excited singlet state is prolonged to the nanosecond timescale; this allows for greater efficiency of fluorescence and increases the probability for intersystem crossing to the triplet state necessary for singlet oxygen mediated photochemical reactions (Wagner S J et al. Transfusion In press; Yamamoto N et al. Nucleic Acids Symp Ser. 1993; 29:83-4; Yamamoto N et al.; U.S. Pat. No. 6,022,961, 2000; Okamoto T et al. U.S. Pat. No. 6,242,477 B1, 2001). TP is a potent photosensitizer against a broad spectrum of viruses and bacteria. Unfortunately, TP also exhibits strong affinity to RBC membranes. Although photoinduced hemolysis from membrane bound TP could almost be reduced to background control levels by adding a competitive membrane binding inhibitor, dipyridamole, this approach still required the addition of two drugs to RBCs (Wagner S J et al. Transfusion 2005; 45:752-60).
There remains a need in the art for methods and materials capable of initiating singlet oxygen mediated photochemical reactions but which have binding specificity for pathogenic components to the exclusion of RBCs and/or other clinically significant components.